PROJECT SUMMARY The RNA m7G cap has been recognized as a capstone in RNA metabolism in eukaryotes, thanks to decades of research that uncovered the mechanisms underlying its deposition, removal, and impacts on gene expression. However, recent discoveries showing that the m7G cap is not the only RNA cap indicate that our knowledge of RNA metabolism is far from complete. Nicotinamide adenine diphosphate (NAD+) has recently emerged as an RNA cap in bacteria, yeast, and humans, and our preliminary studies show that this cap is widespread in the model plant Arabidopsis; thus, NAD+ maybe a universal RNA cap in life. Existing evidence points to a dynamic nature of this RNA modification, as enzymes that deposit and remove the NAD+ cap have been identified in bacteria, humans, and Arabidopsis. The potentially dynamic nature of this RNA modification points to its as yet unknown regulatory functions in gene expression and biological processes. As NAD+ serves critical functions in cellular redox and energy homeostasis, it is possible that NAD+ capping/decapping in RNA is both regulated by and impacts cellular redox and metabolic homeostasis. Despite its potential importance, our knowledge of the NAD+ cap is at most rudimentary. The project seeks to understand the biology of the RNA NAD+ cap using the Arabidopsis model. Based on preliminary studies that documented the existence of NAD+-capped RNAs, implicated their translational status and revealed potential decapping enzymes, the project interrogates how the NAD+ cap is deposited and removed, how the NAD+ cap impacts gene expression, and what biological processes are regulated by RNA NAD+-capping/decapping. The sophisticated molecular and genetic resources in the Arabidopsis model not only allow for the understanding of this universal RNA modification in one domain of life, but also offer advantages of studying this RNA modification in an intact, multicellular life with relative ease. Findings on the RNA NAD+ cap from this project may have far-reaching impacts in agriculture and medicine.